The present invention relates to mutant liquefying xcex1-amylases which have the optimum pH in an alkaline range and excellent resistance to oxidizing agents and are particularly useful as enzymes for detergents comprising an oxidizing agent, and genes thereof.
Various enzymes are incorporated into detergents for the purpose of enhancing detergency, and it is considered to incorporate an xcex1-amylase against, for example, starch smears. However, the xcex1-amylase incorporated into a detergent must be an alkaline xcex1-amylase, since the detergent comprises a surfactant, and the pH of a detergent solution is in an alkaline range.
By the way, detergents, in which an oxidation bleaching component is incorporated to expect not only detergency against dirt, but also a bleaching action, have been recently marketed. It is considered that the xcex1-amylase is also incorporated into such an oxidation bleaching agent-containing detergent. However, the usual xcex1-amylase is easy to inactivate in the presence of an oxidizing agent and has hence been unable to be incorporated into the oxidation bleaching agent-containing detergent.
Researches have been made to impart resistance to oxidizing agents to such an xcex1-amylase. More specifically, WO 94/02597 has provided oxidizing agent-resistant mutant proteins by substituting a non-oxidizing amino acid, particularly, leucine (Leu), threonine (Thr) or glycine (Gly) for a methionine residue of an xcex1-amylase derived from B. licheniformis. WO 94/18314 and WO 96/30481 have reported that when Met residues corresponding to the 197-position and 15-position among methionine residues in the same enzyme as described above are replaced by, in particular, Ala, Ile or Thr and Leu, Thr, Asp, Ser, Val or Ile, respectively, the resistance to oxidizing agents and heat stability of the enzyme are enhanced. However, these mutant xcex1-amylases are all enzymes having the optimum pH in a neutral to acidic range, and there has thus been a demand for development of an alkaline xcex1-amylase having a higher optimum pH for the purpose of using in detergents.
On the other hand, as a technique for imparting resistance to oxidizing agents to an alkaline xcex1-amylase, WO 96/23873 has only reported oxidizing agent-resistant mutant xcex1-amylases obtained by modifying SEQ ID NO:1 (NCIB 12512) encoding an xcex1-amylase. According to WO 96/23873, taking into account the results of the xcex1-amylase derived from B. licheniformis (WO 94/18314), and the like, it is described that when Met corresponding to the 202-position in FIG. 2 of NCIB 12512 is replaced by Leu, Phe, Ala, Thr, Val or Ser, the resultant xcex1-amylase mutant becomes oxidizing agent-resistant to a treatment with 200 mM H2O2. It has however been reported that since its resistance to oxidation is insufficient, Arg at the 181-position and Gly at the 182-position are deleted in addition to this mutation (Suzuki et al., J. Biol. Chem., 264, 18933-18938, 1989) to more stabilize the mutant. However, the half-lives (txc2xd) of enzymatic activity of the mutant xcex1-amylases obtained in such a manner are only in ranges of 10 to 20 minutes for the former and 10 to 30 minutes for the latter, and so they are not satisfactory as enzymes for incorporating into detergents for both resistance to oxidizing agents and lastingness thereof.
Accordingly, it is an object of the present invention to provide an xcex1-amylase which has the optimum pH in an alkaline range and lasting and strong resistance to oxidizing agents, a gene thereof, and a detergent composition comprising such an xcex1-amylase.
In view of the foregoing circumstances, the present inventors have paid attention to an enzyme produced by Bacillus sp. KSM-AP 1378 (WO 94/26881), which is a sort of liquefying alkaline xcex1-amylase, and carried out various investigations. As a result, it has been found that when at least one methionine residue at the 202-position or a position homologous thereto in the amino acid sequence set forth in SEQ ID NO:1 is deleted or replaced by another arbitrary amino acid residue, the resultant xcex1-amylase mutant comes to have strong and lasting resistance to oxidizing agents and has an excellent amylase activity in an alkaline pH range, thus leading to completion of the present invention.
According to the present invention, there is thus provided a mutant xcex1-amylase having an amino acid sequence obtained by making deletion or replacement by another arbitrary amino acid residue of a methionine residue at the 202-position or a position homologous thereto in the amino acid sequence set forth in SEQ ID NO:1, which constitutes a liquefying alkaline xcex1-amylase, or in an amino acid sequence having a homology of at least 95.2% to said amino acid sequence.
According to the present invention, there is also provided a gene encoding the mutant xcex1-amylase.
According to the present invention, there is further provided a detergent composition comprising the mutant xcex1-amylase.